Project Summary Surgical meshes used to provide mechanical support and contain tissue or organ prolapse (e.g., hernia, pelvic/vaginal extrusion) often incite complications resulting from excessive foreign body reaction (e.g., mesh erosion, infection, chronic inflammation, tissue stiffening due to excessive fibrosis). This is exemplified in reconstructive surgery to treat pelvic organ prolapse (POP), a condition wherein pelvic organs protrude through the vagina due to loss of structural support. In addition, chronic extracellular matrix (ECM: collagen, elastin) breakdown and the inability of adult cells to regenerate and repair elastic fibers are associated with POP, as we have confirmed in a lysyl oxidase like -1 knockout (LOXL1 KO) mouse model. These pathophysiologic changes also continue following mesh implantation to cause vaginal scarring, mesh shrinkage and erosion, and POP relapse. The goal of this project is to investigate an adjuvant ECM regenerative nano- pharmacotherapy to both reduce mesh complications and reverse ECM pathophysiology in the mesh-tissue complex at the vaginal wall in a mouse model of POP to improve outcomes of surgical mesh implantation. We have developed biodegradable polyethylene glycol (PEG)-polylactic-co-glycolic acid (PLGA) Nano Particles (NPs) which provide predictable, sustained and steady-state release of doxycycline (DOX), a matrix metalloprotease (MMP)-inhibitory drug that also has anti-microbial properties. Our NPs present pendant functional groups that inhibit MMPs independent of the released DOX, and also stimulate elastic matrix neoassembly. We showed that in the 0.1-10 ?g/ml dose range at which the DOX is released from NPs, the drug uniquely provides both pro-elastogenic and anti-MMP stimuli, and that these are linked to DOX inhibition of c-Jun N-terminal kinase (JNK), a protein kinase activated during inflammation. JNK inhibition upregulates transforming growth factor ? (TGF-?1), which we have shown separately to stimulate elastogenesis in cultures of non-epithelial vaginal cells (NEVCs) established from our LOXL1 KO model. Since JNK promotes connective tissue growth factor (CTGF), which is essential for fibroblast activation and fibrosis, we expect that DOX inhibition of JNK will also reduce fibrosis. In this study, we propose to clarify the mechanisms underlying these multimodal effects of DOX in the context of treating POP. We will test a hypothesis that localized & sustained release of DOX from matrix-regenerative NPs in the vaginal wall given at the time of surgical mesh implantation will provide long-term pro-elastogenic, anti-MMP stimuli, and attenuate fibrosis and infections, towards reducing mesh complications and improving POP treatment outcomes. Aim 1 will investigate effects of DOX-NPs on JNK inhibition & downstream pro-elastogenic, anti-proteolytic & anti-fibrotic effects in NEVC cultures established from LOXL1 KO mice with POP. Aim 2 will investigate efficacy of adjuvant DOX-NP therapy for improving mesh implantation outcomes in LOXL1 KO mice with POP. This study will provide feasibility data for future studies using our paradigm-shifting NP approach to improve outcomes of surgical mesh implantation, including those used clinically for POP.